The invention relates to a method of chromatographic isolation for non-glyceride components particularly squalenes, carotenes, vitamin E (sometimes abbreviated as Vit. E) sterols and/or the like, from crude palm oil, palm oils products and/or by-products, vegetable oils and/or the like non-glyceride components-comprising compounds.
Crude palm oil contains about 5% of xe2x80x9cnon-glyceride componentsxe2x80x9d which include carotenoids, tocols (tocopherols and tocotrienols), sterols and squalene. The carotenoids, present at 500-700 ppm and comprising mainly xcex1 and xcex2 carotenes, are important constituents with pro-vitamin A activity, possible antitumor formation properties, and other physiological activities. The tocols which are Vitamin E and also natural anti-oxidants, are present at approximately 600 to 1000 ppm in crude palm oil; the major component being the gamma-tocotrienol which has recently been found to have anti-cancer properties besides its known anti-oxidant activity. Tocotrienol has been found to lower blood cholesterol. The sterols consisting mainly of xcex2-sitosterol, stigmasterol and campesterol, provide raw materials for steroid intermediates and drugs. xcex2-Sitosterol also possesses hypocholesterolemic effect. Squalene is an important ingredient for cosmetics. It also shows beneficial physiological properties.
Several methods have been developed to extract these valuable compounds. In the case of the carotenoids, the known methods can be classified as follows:
(i) Extraction by saponification e.g. British Patent 567,682; U.S. Pat. Nos. 2,460,796; 2,440,029; 2,572,467; 2,652,433.
(ii) Iodine method
(iii) Urea process
(iv) Extraction using Fuller""s earth or activated carbon, e.g. British Patent 691,924; British Patent 1,562,794; U.S. Pat. No. 2,484,040.
(v) Extraction by selective solvents e.g. U.S. Pat. No. 2,432,021.
(vi) Molecular Distillation.
In the saponification method (i) the oil is saponified to give soap, glycerol and a non-saponifiable fraction containing carotenes.
In the iodine method (ii), the iodine is added to a solution of palm oil in petroleum ether, an insoluble precipitate of carotene di-iodide is formed. The iodo-compound when treated with sodium thiosulphate however yields iso-carotene or dehydro-carotene which has no biological activity.
With the urea method (iii), the triglycerides are broken down to fatty acids and methyl esters which then form insoluble compounds with urea and thiourea, leaving the carotenoids in the remaining liquid.
Extraction of carotenes using adsorbents has been carried out using Fuller""s earth and activated carbon (method iv). However, the extraction of the carotenes from the earth gives oxidised or isomerised products of carotenes. Carotene is concentrated six times in the extract.
Extraction of carotenes by selective solvents (method v) has been carried out using propane or furfural. The carotene is concentrated (three times that of the original oil) in the furfural phase.
By method (vi) carotenes can also be obtained by molecular distillation (10xe2x88x923-10xe2x88x924 mm Hg). Fractions collected at 230xc2x0 C. have a carotene content of about five times that of the original oil.
To-date, there has not been any method which discloses the use of supercritical fluid in the adsorption/desorption chromatography isolation/separation of the non-glyceride components (i.e. carotenoids, tocols, sterols and squalene) from plant source such as CPO (crude palm oil), palm oil products and/or by-products, vegetable oils and fats and/or the like non-glyceride components-comprising compounds.
All known method previously disclosed in the adsorption/desorption cromatographic separation solely involve the use of solvents (which are costly and hazardous). Thus, there is a need to provide a separation and/or an isolation process, which avoids or discourages the sole use of solvents and consequently rendering it xe2x80x9cnon-hazardousxe2x80x9d for recovering these non-glyceride components.
In accordance with the present invention, there is provided a method of chromatographic isolation for non-glyceride components (carotenes, vitamin E [sometimes abbreviated as Vit. E], sterols, squalene) from natural oils and fats, wherein the method comprises the use of supercritical fluid (such as SC-CO2) in combination with adsorbents (such as silica gel or C18 reverse phase silica gel). The present invention may be subjected directly and/or indirectly to
(a) crude vegetable oil esters (via catalytic alcoholic esterification/transesterification of vegetable oils) or
(b) concentrate (through removal of the bulk of the esters from (a) via vacuum distillation including molecular distillation) or
(c) the unsaponifiable matters of the concentrate in (b) (through saponification process)
High concentration of carotenes, vitamin E, sterols and squalene can be obtained by monitoring supercritical fluid process conditions such as temperature between 30xc2x0 C.-100xc2x0 C., pressure from 50 kg/cm2 to 600 kg/cm2, with and without an to entrainer (co-solvent such as alcohols).
A main advantage of the invention lies in the use of liquified gas at supercritical conditions, hence avoiding or reducing the prior requirements for hazardous solvents.
According to the present invention there is provided a method of chromatographic isolation for xe2x80x9cminor non-glyceride componentsxe2x80x9d (including carotenoids, tocols, sterols and squalene) from CPO (crude palm oil), palm oil products and/or by-products, vegetable oil and/.or the like non-glyceride components-comprising compounds, said method comprising the following steps for three different routes as described below:
Route (a)
(i) Esterifying the free fatty acid component of the non-glyceride components-comprising compounds with one or more monohydric alcohols to form an esterified compound with a very low free fatty acid content;
(ii) Trans-esterifying the glyceride components with one or more monohydirc alcohols to convert into monoesters;
(iii) Introducing the non-glyceride components-comprising compounds (i.e. esters from steps (i) and (ii) above) to allow the adsorption of non-glyceride components (carotenoids, tocols, sterols and squalene) onto a selective adsorbent; and subsequently
(iv) Desorbing the non-glyceride components from the adsorbent, wherein the adsorption/desorption of the non-glyceride components in steps (iii)-(iv) is carried out under a supercritical fluid environment.
Route (b)
(i) Esterifying the free fatty acid components of the non-glyceride components-comprising compounds with one or more monohydric alcohols to form an esterified compound with a very low free fatty acid content;
(ii) Trans-esterifying the glyceride components with one or more monohydirc alcohols to convert into monoesters;
(iii) Removing bulk of the esters in step (i) and (ii) by vacuum distillation including molecular distillation to yield carotene concentrate of 1-8%;
(iv) Introducing the non-glyceride components-comprsing compounds (i.e. the carotene concentrate from step (iii) above) to allow the adsorption of non-glyceride components (carotenoids, tocols, sterols and squalene) onto a selective adsorbent; and subsequently
(v) Desorbing the non-glyceride components from the adsorbent, wherein the adsorption/desortion of the non-glyceride components in steps (iv)-(v) is carried out under a supercritical fluid environment.
Route (c)
(i) Esterfying the free fatty acid component of the non-glyceride components-comprising compounds with one or more monohydirc alcohols to form an esterifed compound with a very low free fatty acid content;
(ii) Trans-esterdifying the glyceride components with one or more monohydric alcohols to convert into monoesters;
(iii) Removing bulk of the esters in step (i) and (ii) by vacuum distillation including molecular distillation to yield carotene concentrate of 1-8%.
(iv) Saponifying the carotene concentrate in step (iii) to yield unsaponifiable matters;
(v) Introducing the non-glyceride components-comprising compounds to allow the adsorption of non-glyceride components (carotenoids, tocols, sterols and squalene) onto a selective adsorbent; and subsequently
(vi) Desorbing the non-glyceride components from the adsorbent, wherein the adsorption/desorption of the non-glyceride components in steps (v)-(vi) is carried out under a supercritical fluid environment.
Preferably, the esterification of step (i) above is carried out employing
(a) a solid alkali metal bisulphate or
(b) a sulphate acid strongly-acidic ion-exchange resin.
Preferably, the transesterification of step (ii) is carried out employing a basic catalyst.
Alternatively, both the esterification and transesterification are carried out using an enzyme; for example candida rugosa and lipase. 1 to 20% by weight of catalyst may be employed depending upon the weight of the free fatty carboxylic to acid moiety present in the non-glyceride components-comprising compounds.
Preferably, the monohydric alcohols used in the esterification of step (i) or transesterification of step (ii) comprise one or more C1 to C8 alcohols, most preferably methanol.
Preferably, the selective adsorbent for the adsorption of the non-glyceride components comprise silica gel and/or C18 reverse phase silica gel. The adsorbents may be subjected to adsorption of the non-glyceride components present within any one of the following;
(i) the esterified palm oil; or
(ii) the carotene concentrate (1-8%) wherein bulk of the esters have been removed through vacuum distillation or molecular distillation (short path distillation); or
(iii) the unsaponifiable materials by saponifying the carotene concentrate of (ii).
The solvents employed for the present method comprises;
(i) Supercritical fluid, e.g. SC-CO2 (100%), and/or
(ii) Supercritical fluid (e.g. SC-CO2) in combination with an entrainer (solvent such as alcohol)
Carotene, tocols, sterols and squalene can be isolated with  greater than 50% purity in a single run by varying pressure from 50 kg/cm2-600 kg/cm2 at temperatures 30xc2x0 C.-100xc2x0 C.
It shall therefore be illustrated, by means of the following examples, that supercritical fluid, applied to solid adsorbents such as silica gel and/or C18 reverse phase silica gel, provides a very satisfactory means for isolating non-glyceride components (including carotenoids, tocols, sterols and squalene) from CPO (crude palm oil), palm oil products and/or by-products, vegetable oil and/or the like non-glyceride components-comprising compounds.